This application claims priority from International Application No. PCT/EP98/02483, filed Apr. 27, 1998 and German Application No. 197 17 656.9, filed Apr. 25, 1997. Each of the foregoing applications, patents and publications and all documents cited or referenced therein (xe2x80x9capplication cited documentsxe2x80x9d) and all documents cited or referenced in this specification (xe2x80x9cherein cited documentsxe2x80x9d) are hereby incorporated herein by reference.
Not applicable.
The present invention relates to the subunit CHLD of plant magnesium chelatase (Mg chelatase), to DNA sequences encoding the subunit CHLD of plant Mg chelatase, to processes for preparing the subunit CHLD of plant Mg chelatase, to processes for determining the activity of plant Mg chelatase, and transgenic plants which have been transformed with the Mg chelatase DNA according to the invention.
Being photosystem cofactors, chlorophylls play a role in the conversion of light into chemical energy and are thus required for plant growth and survival.
Magnesium is incorporated into porphyrin during chlorophyll biosynthesis with the aid of a membranexe2x80x94associated enzyme, namely Mg chelatase, which is composed of several subunits.
It has already been disclosed that bacterial Mg chelatase is composed of three subunits (D, H and I), whose corresponding gene sequences are termed bchD, bchH and bchI (Burke et al. (1993), J. Bacteriol. 175, 2414-2422; Coomber et al. (1990), Mol. Microbiol. 4, 977-989; Gibson et al. (1995), Proc. Natl. Acad. Sci. USA, 92, 1941-1844; Jensen et al. (1996), J. Biol. Chem. 271, 16662-16667).
As regards plant Mg chelatases, two subunits have been described to date which seem to correspond to the bacterial Mg chelatase subunits bchH and bchI (Koncz et al., (1990), EMBO J. 9, 1337-1346; Hudson et al., (1993), EMBO J. 12, 3711-3719; Eibson et al., (1996), Plant Physiol. 121, 61-71). It is not known as yet which other subunits participate in the structure of plant Mg chelatase. No enzyme activity was observed with the two known plant subunits CHLI and CHLH, neither alone nor together with the known bacterial subunits of type D (CHLD and BCHD).
Due to their key position in chlorophyll biosynthesis, plant Mg chelatase is a radically new starting point for developing a novel generation of herbicidal compounds with highly specific activity. In addition, the vitality and/or growth of phototrophic uni- and multicellular organisms, in particular bacteria, algae and plants, can be controlled to a high degree by influencing gene expression (suppression, overexpression) of the natural or modified (for example genetically engineered) expression products of Mg chelatase or else by specific Mg chelatase inhibitors.
The enzymatic activity of plant Mg chelatase was originally measured on intact chloroplasts (Castelfranco et al., (1979) Arch. Biochem. Biophys. 192, 592-598; Fuesler et al. (1982) Plant Physiol. 69,421-423). Since then, the activity was also determined on disrupted plastids (Walker et al. (1991) Proc. Natl. Acad. Sci. 88, 5789-5793) and subplastid membrane fractions (Lee, et al. (1992). Plant Physiol. 99,1134-1140).
Surprisingly, there has now been found a DNA which encodes a subunit of the enzyme plant Mg chelatase. The DNA will subsequently be termed chlD, and the amino acid sequence CHLD.
Moreover, it has been found that a subunit CHLD of plant Mg chelatase together with the subunits CHLI and CHLH is, surprisingly, suitable for reconstituting a funtionally intact, i.e. enzymatically active, plant Mg chelatase, so that the plant Mg chelatase subunit CHLD according to the invention provides novel test methods (in vivo and in vitro) for plant Mg chelatase activity.